Activated Oxytocin Neurons in the PVN-DVC Pathway in Asthmatic Rats

Abstract

Asthma is a heterogeneous disease, and the central nervous system (CNS) also participates in the pathogenesis of asthma. We previously reported the amygdala might regulate asthmatic attacks via projecting to the paraventricular hypothalamic nucleus (PVN). The dorsal vagal complex (DVC) is a crucial region that modulates respiratory. This study aimed to observe the activity in both PVN and DVC and the connection between PVN and DVC in asthmatic rats. Immunohistochemistry was conducted to observe the changes in Fos and oxytocin (OT) expression. Retrograde tracing using wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and double immunohistochemistry for OT and Fos was used to observe the HRP/OT/Fos positive neurons distribution in the PVN. The results showed that during an asthma attack, the Fos positive neurons increased in both PVN and DVC over time. The expression of OT positive neurons in PVN showed a similar trend in parallel to the c-Fos positive neurons in PVN. The HRP retrograde-labeled neurons were densely distributed in the medial and lateral subnucleus in the PVN. OT⁺/HRP⁺ and Fos⁺/OT⁺/HRP⁺ accounted for 18.14%, and 2.37% of HRP-labeled neurons, respectively. Our study showed PVN and DVC were activated and the expression of OT positive neurons in PVN were increased over time during an asthma attack. The existence of connection between PVN and DVC suggested the OT neurons in PVN might project to DVC which might be involved in the pathogenesis of asthma.

Keywords: asthma; dorsal vagal complex; oxytocin; paraventricular nucleus; pathway.

FIGURE 1

(A) Histopathological changes of lung tissues in asthma and control rats. (B) Percentage of cells in BALF in asthma and control rats. (C) Changes of RF, V_T, and MVV before and after challenge. (D) T_E/T_I, R_aw, and C_dyn in asthma and control rats. (E) EMGdi frequency and IDEA in asthma and control rats. (F) Changes of P_aO₂, P_aCO₂, and S_aO₂ in control and asthma (5, 10, and 30 min) rats. After the rats were challenged, the lung function was deteriorated and the relevant indicators of ventilation function were significantly changed. Meanwhile, the airway inflammation, especially the eosinophils in BALF, was significantly increased. At the same time, as the duration of an asthma attack increased, arterial oxygen saturation was decreased. *p<0.05, **p<0.01, ***p<0.001, and #p>0.05, respectively. n = 6 per group. BALF, bronchoalveolar lavage fluid; RF, respiratory frequency; V_T, tidal volume; MVV, minute ventilation volume; T_E/T_I, expiratory time course/inspiratory time course ratio; R_aw, airway resistance; C_dyn, dynamic pulmonary compliance; EMGdi frequency, frequency of diaphragmatic electric activity; IDEA, integrated diaphragmatic electrical activity.

FIGURE 2

(A) Fos expressions in PVN in asthma and control rats. (B) Counts of Fos-positive neurons in PVN in control and asthma (30, 60, 90, and 120 min) rats. (C) Fos expressions in DVC in asthma and control rats. (D) Counts of Fos-positive neurons in DVC in control and asthma (30, 60, 90, and 120 min) rats. The nucleus of the Fos-positive neurons was brownish yellow staining, and Fos expressions increased gradually in the 30, 60, and 90 min and peaked at the 120 min. ***p<0.001. n = 6 per group. PVN, paraventricular nucleus; DVC, dorsal vagal complex; 3V, third ventricle; cc, central canal.

FIGURE 3

(A) OT immunoreactive substance in PVN in asthma and control rats. (B) Mean density of OT immunoreactive substance in PVN in control and asthma (30, 60, 90, and 120 min) rats. The OT immunoreactive substance was brown staining, and OT expressions increased and peaked in the 90 and 120 min. ***p<0.001, and #p>0.05, respectively. n = 6 per group. PVN, paraventricular nucleus; 3V, third ventricle.

FIGURE 4

(A) The distribution of HRP-labeled neurons in the PVN. (B) The microinjection zone of HRP in DVC. HRP injection zone could be observed on one side of DVC, and HRP retrograde-labeled neurons were densely distributed in the medial subnucleus and the lateral subnucleus of PVN and scattered in the dorsal subnucleus. PVN, paraventricular nucleus; DVC, dorsal vagal complex; 3V, third ventricle; 4V, fourth ventricle; cc, central canal; HRP, horseradish peroxidase.

FIGURE 5

(A) HRP/Fos/OT labeled neurons in PVN. (B) Seven different immuno-positive neurons (red arrow) distribution in PVN in asthma rats. (C) The ratio of different types of HRP labeled neurons. OT labeled neurons, Fos labeled neurons, HRP labeled neurons, HRP/OT labeled neurons, HRP/Fos labeled neurons, Fos/OT labeled neurons, and HRP/Fos/OT labeled neurons were observed in PVN. HRP, horseradish peroxidase; PVN, paraventricular nucleus.

FIGURE 6

The scheme of the possible role of PVN-DVC circuit on modulating respiratory response during an asthma attack. During asthma attack, neurons in PVN and DVC were activated, and OT expressions in PVN were increased. The OT neurons in PVN may project to DVC to regulate the asthma attack.